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Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.

Plafker SM, Plafker KS, Weissman AM, Macara IG - J. Cell Biol. (2004)

Bottom Line: We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11.This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes.Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. scott-plafker@ouhsc.edu

ABSTRACT
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

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Class III E2s can access the nucleus by the same import pathway. BHK cells were microinjected in the cytoplasm with TRITC-labeled dextran (Inj marker; 1mg/ml), GFP-UbcH6-H6 (A), or GFP-UBE2E2-H6 (B), and a 20-molar excess of GST-UbcM2 (C145A) (a, b, e, and f) or GST-UbcM2 (wt) (c, d, g, and h). After injection, cells were incubated for 15 min at 37°C and analyzed live by fluorescence microscopy. 50–75 cells were injected for each condition. Bar, 10 μm. (C) Cells injected with GST-UbcM2 (C145A) (i–k) or GST-UbcM2 (wt) (l–n) were incubated for 30 min at 37°C and fixed. The localization of the GST fusions was determined by indirect immunofluorescence using an anti-GST antibody and a FITC-conjugated secondary antibody. DNA was stained with DAPI to mark the nuclear compartment for each cell.
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fig5: Class III E2s can access the nucleus by the same import pathway. BHK cells were microinjected in the cytoplasm with TRITC-labeled dextran (Inj marker; 1mg/ml), GFP-UbcH6-H6 (A), or GFP-UBE2E2-H6 (B), and a 20-molar excess of GST-UbcM2 (C145A) (a, b, e, and f) or GST-UbcM2 (wt) (c, d, g, and h). After injection, cells were incubated for 15 min at 37°C and analyzed live by fluorescence microscopy. 50–75 cells were injected for each condition. Bar, 10 μm. (C) Cells injected with GST-UbcM2 (C145A) (i–k) or GST-UbcM2 (wt) (l–n) were incubated for 30 min at 37°C and fixed. The localization of the GST fusions was determined by indirect immunofluorescence using an anti-GST antibody and a FITC-conjugated secondary antibody. DNA was stained with DAPI to mark the nuclear compartment for each cell.

Mentions: The coimmunoprecipitation data predict that all three human class III E2s can localize to the nucleus by accessing the importin-11 pathway. We reasoned that the nuclear import of any of these E2s should be specifically prevented by saturating the importin-11 pathway with an excess of a second class III enzyme. To test this prediction, BHK cells were injected in the cytoplasm with GFP-UbcH6-H6 or GFP-UBE2E2-H6 and a 20-molar excess of either GST-UbcM2(C145A) or GST-UbcM2(wt). After a 15-min incubation at 37°C, the cells were analyzed live by fluorescence microscopy. The GFP-E2 fusions localized efficiently to the nucleus in the presence of the GST-UbcM2(C145A) competitor (Fig. 5 Ab; Fig. 5 Bf), but their import was effectively competed by GST-UbcM2(wt) (Fig. 5 Ad; Fig. 5 Bh). Similar results were found using His-S-tagged UbcM2 (wt or C145A) as competitors (Fig. S1 B). The import of both GFP-E2 fusions was also inhibited by coinjecting Ran (Q69L) (unpublished data). When this experiment was done using an excess of GST-UbcH7 as a competitor, both GFP fusions localized efficiently to the nucleus (Fig. S1 C). Therefore, the differential effects of the wt and inactive UbcM2 competitors was not simply a consequence of the wt UbcM2 competitor overwhelming the Ub-charging capacity of endogenous E1 and preventing activation of the GFP-E2s. The localizations of the competitor GST fusions were validated in a separate micro-injection experiment. As expected, GST-UbcM2(C145A) was distributed throughout the cytoplasm (Fig. 5 C, i–k), and GST-UbcM2(wt) accumulated in the nucleus (Fig. 5 C, l–n). Together, these injection data demonstrate that these three human class III E2s can access the nucleus by the importin-11 pathway.


Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.

Plafker SM, Plafker KS, Weissman AM, Macara IG - J. Cell Biol. (2004)

Class III E2s can access the nucleus by the same import pathway. BHK cells were microinjected in the cytoplasm with TRITC-labeled dextran (Inj marker; 1mg/ml), GFP-UbcH6-H6 (A), or GFP-UBE2E2-H6 (B), and a 20-molar excess of GST-UbcM2 (C145A) (a, b, e, and f) or GST-UbcM2 (wt) (c, d, g, and h). After injection, cells were incubated for 15 min at 37°C and analyzed live by fluorescence microscopy. 50–75 cells were injected for each condition. Bar, 10 μm. (C) Cells injected with GST-UbcM2 (C145A) (i–k) or GST-UbcM2 (wt) (l–n) were incubated for 30 min at 37°C and fixed. The localization of the GST fusions was determined by indirect immunofluorescence using an anti-GST antibody and a FITC-conjugated secondary antibody. DNA was stained with DAPI to mark the nuclear compartment for each cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172591&req=5

fig5: Class III E2s can access the nucleus by the same import pathway. BHK cells were microinjected in the cytoplasm with TRITC-labeled dextran (Inj marker; 1mg/ml), GFP-UbcH6-H6 (A), or GFP-UBE2E2-H6 (B), and a 20-molar excess of GST-UbcM2 (C145A) (a, b, e, and f) or GST-UbcM2 (wt) (c, d, g, and h). After injection, cells were incubated for 15 min at 37°C and analyzed live by fluorescence microscopy. 50–75 cells were injected for each condition. Bar, 10 μm. (C) Cells injected with GST-UbcM2 (C145A) (i–k) or GST-UbcM2 (wt) (l–n) were incubated for 30 min at 37°C and fixed. The localization of the GST fusions was determined by indirect immunofluorescence using an anti-GST antibody and a FITC-conjugated secondary antibody. DNA was stained with DAPI to mark the nuclear compartment for each cell.
Mentions: The coimmunoprecipitation data predict that all three human class III E2s can localize to the nucleus by accessing the importin-11 pathway. We reasoned that the nuclear import of any of these E2s should be specifically prevented by saturating the importin-11 pathway with an excess of a second class III enzyme. To test this prediction, BHK cells were injected in the cytoplasm with GFP-UbcH6-H6 or GFP-UBE2E2-H6 and a 20-molar excess of either GST-UbcM2(C145A) or GST-UbcM2(wt). After a 15-min incubation at 37°C, the cells were analyzed live by fluorescence microscopy. The GFP-E2 fusions localized efficiently to the nucleus in the presence of the GST-UbcM2(C145A) competitor (Fig. 5 Ab; Fig. 5 Bf), but their import was effectively competed by GST-UbcM2(wt) (Fig. 5 Ad; Fig. 5 Bh). Similar results were found using His-S-tagged UbcM2 (wt or C145A) as competitors (Fig. S1 B). The import of both GFP-E2 fusions was also inhibited by coinjecting Ran (Q69L) (unpublished data). When this experiment was done using an excess of GST-UbcH7 as a competitor, both GFP fusions localized efficiently to the nucleus (Fig. S1 C). Therefore, the differential effects of the wt and inactive UbcM2 competitors was not simply a consequence of the wt UbcM2 competitor overwhelming the Ub-charging capacity of endogenous E1 and preventing activation of the GFP-E2s. The localizations of the competitor GST fusions were validated in a separate micro-injection experiment. As expected, GST-UbcM2(C145A) was distributed throughout the cytoplasm (Fig. 5 C, i–k), and GST-UbcM2(wt) accumulated in the nucleus (Fig. 5 C, l–n). Together, these injection data demonstrate that these three human class III E2s can access the nucleus by the importin-11 pathway.

Bottom Line: We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11.This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes.Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. scott-plafker@ouhsc.edu

ABSTRACT
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

Show MeSH